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Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organ-isms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible func-tion of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially represent-ing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

Enzymatic proteins with a metallo-beta-lactamase (MBL) fold have been essentially studied in bacteria for their activity on beta-lactam antibiotics. However, the MBL fold is ancient and highly conserved, and these proteins are capable of cleaving a broad range of substrates. It has recently been shown that MBLs are present in a wide array of cellular organisms, including eukaryotes and archaea. We show here that Tupanvirus deep ocean, a giant virus, also encodes a protein with a MBL fold. Phylogeny showed its clustering with transfer ribonucleases (RNases) and the presence of orthologs in other giant viruses, mainly those harboring the largest sets of translation components. In addition, it suggests an ancient origin for these genes and a transfer between giant viruses and Acanthamoeba spp., a host of many giant viruses. Biologically, after its expression in Escherichia coli, the tupanvirus protein was found to hydrolyse nitrocefin, a chromogenic beta-lactam. We also observed an hydrolysis of penicillin G (10 μg/mL) and detected the metabolite of penicillin G hydrolysis, benzylpenilloic acid. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, we tested the degradation of single-stranded DNA, double-stranded DNA, and RNAs, and observed a strong activity on RNAs from seven bacteria with G+C varying from 42% to 67%, and from Acanthamoeba castellanii, the tupanvirus host. This was not inhibited by sulbactam or ceftriaxone. RNase activity was estimated to be 0.45±0.15 mU/mg using a fluorescence-based assay. Our results still broaden the range of hosts of MBL fold proteins and demonstrate that such protein can have dual beta-lactamase/nuclease activities. We suggest that they should be annotated according to this finding to avoid further confusion.

The aldehyde dehydrogenases (ALDHs) are a superfamily of multimeric enzymes which catalyse the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the reduction of their cofactor, NAD or NADP, into NADH or NADPH. At present, the only known structures concern NAD-dependent ALDHs. Three structures are available in the Protein Data Bank: two are tetrameric and the other is a dimer. We solved by molecular replacement the first structure of an NADP-dependent ALDH isolated from Streptococcus mutans, in its apo form and holo form in complex with NADP, at 1.8 and 2.6 A resolution, respectively. Although the protein sequence shares only approximately 30 % identity with the other solved tetrameric ALDHs, the structures are very similar. However, a large local conformational change in the region surrounding the 2′ phosphate group of the adenosine moiety is observed when the enzyme binds NADP, in contrast to the NAD-dependent ALDHs. Structure and sequence analyses reveal several properties. A small number of residues seem to determine the oligomeric state. Likewise, the nature (charge and volume) of the residue at position 180 (Thr in ALDH from S. mutans) determines the cofactor specificity in comparison with the structures of NAD-dependent ALDHs. The presence of a hydrogen bond network around the cofactor not only allows it to bind to the enzyme but also directs the side-chains in a correct orientation for the catalytic reaction to take place. Moreover, a specific part of this network appears to be important in substrate binding. Since the enzyme oxidises the same substrate, glyceraldehyde-3-phosphate (G3P), as NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the active site of GAPDH was compared with that of the S. mutans ALDH. It was found that Arg103, Arg283 and Asp440 might be key residues for substrate binding.

International audience; The study of the human virome is still in its infancy, especially with regard to the viral content of the blood of people who are apparently disease free. In this study, the genome of a new giant virus that is related to the amoeba-infecting pathogen Marseillevirus was recovered from donated blood, using high-throughput sequencing. Viral antigens were identified by an immunoconversion assay. The virus was visualized with transmission electron microscopy and fluorescence in situ hybridization and was grown in human T lymphocytes. Specific antibody reactions were used to identify viral proteins in blood specimens from polymerase chain reactive-positive donors. Finally, we tested 20 blood specimens from additional donors. Three had antibodies directed against this virus, and 2 had circulating viral DNA. This study shows that giant viruses, which are missed by the use of ultrafilters, are part of the human blood virome. The putative pathogenic role of giant viruses in humans remains undefined.

International audience; Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of foreign languages.

Most phagocytic protist viruses have large particles and genomes as well as many laterally acquired genes that may be associated with a sympatric intracellular life (a community-associated lifestyle with viruses, bacteria, and eukaryotes) and the presence of virophages. By subculturing Mimivirus 150 times in a germ-free amoebal host, we observed the emergence of a bald form of the virus that lacked surface fibers and replicated in a morphologically different type of viral factory. When studying a 0.40-μm filtered cloned particle, we found that its genome size shifted from 1.2 (M1) to 0.993 Mb (M4), mainly due to large deletions occurring at both ends of the genome. Some of the lost genes are encoding enzymes required for posttranslational modification of the structural viral proteins, such as glycosyltransferases and ankyrin repeat proteins. Proteomic analysis allowed identification of three proteins, probably required for the assembly of virus fibers. The genes for two of these were found to be deleted from the M4 virus genome. The proteins associated with fibers are highly antigenic and can be recognized by mouse and human antimimivirus antibodies. In addition, the bald strain (M4) was not able to propagate the sputnik virophage. Overall, the Mimivirus transition from a sympatric to an allopatric lifestyle was associated with a stepwise genome reduction and the production of a predominantly bald virophage resistant strain. The new axenic ecosystem allowed the allopatric Mimivirus to lose unnecessary genes that might be involved in the control of competitors.

“Nanobacteria” are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of “nanobacteria” susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of “nanobacteria” as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call “nanons.” The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor., Author Summary In the last decade, the exact nature of nanobacteria was one of the most controversial of scientific questions. An audacious theory proposed the existence of nanobacteria, initially discovered in Italian hot spring deposits, as a new life form responsible for a wide range of diseases in humans, thus qualifying them as new agents of emerging infectious diseases. The community of microbiologists remained therefore skeptical about the fact that such structures, 100 times smaller than bacteria and highly resistant to heat and other treatments that would normally kill the latter, could be living entities fully capable of self-replication. Other scientists wondered if they might be an unusual form of crystal rather than micro-organisms. The comprehensive characterization of nanobacteria was the focus of our study. Our results definitively ruled out the existence of nanobacteria as living entities and revealed that they correspond to self-propagating mineral-fetuin complexes that we called “nanons.”

The proteome of Rickettsia felis, an obligate intracellular bacterium responsible for spotted fever, was analyzed using two complementary proteomic approaches: 2-DE coupled with MALDI-TOF, and SDS-PAGE with nanoLC-MS/MS. This strategy allowed identification of 165 proteins and helped to answer some questions raised by the genome sequence of this bacterium. We successfully identified potential virulence factors including two putative adhesins, four proteins of the type IV secretion system, four Sca autotransporters, four components of ABC transporters, some R. felis-specific proteins, and one antitoxin of the toxin-antitoxin system. Notably, the antitoxin was the first to be identified in intracellular bacteria. Only one protein containing rickettsia palindromic repeats was found, whereas none of the split genes, transposases, or tetratricopeptide/ankyrin repeats were detectably expressed. Comparison of the protein expression profiles of R. felis and 23 other bacterial species according to functional categories showed that intracellular bacteria express more proteins related to translation, especially ribosomal proteins. However, the remaining bacteria express more proteins related to energy production and carbohydrate/amino acid metabolism. In conclusion, this study reveals R. felis virulence factor expression and highlights the unique protein expression profile of intracellular bacteria.

Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 “ORFans.” As demonstrated by both Western blotting and immunogold staining, some of these “ORFans,” which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.

The rickettsial membrane proteins that promote their uptake by eukaryotic host cells are unknown. To identify rickettsial ligand(s) that bind host cell surface proteins, biotinylated epithelial cells were used to probe a nitrocellulose membrane containing rickettsial extracts separated by SDS-PAGE. This overlay assay revealed that two close rickettsial ligands of approximately 32-30 kDa were recognized by host cells. Both proteins were identified using high resolution 2D-PAGE coupled with mass spectrometry analysis. One protein was identified as the C-terminal extremity of rOmpB called the beta-peptide. The second interacting protein was identified as a protein of unknown function encoded by RC1281 and RP828 in Rickettsia conorii and in Rickettsia prowazekii, respectively, that shares strong similarities with other bacterial adhesins. Both proteins are highly conserved within the Rickettsia genus and might play a critical role in their pathogenicity. These data may have important implications for the development of future vaccines against rickettsial infections.

International audience; In this work, we present a comparative two-dimensional (2D) PAGE analysis of Rickettsia conorii and Rickettsia prowazekii. This analysis reveals protein spots that were either unique to or common to both strains, some of them being identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide. The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino acids that contains the active site. The core is wrapped by two long N- and C-terminal extended chains. The catalytic mechanism of the E. coli enzyme has been recently postulated to take place through formation of a sulfenic acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation. In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities. All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol. However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-dependent manner. The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low K(M) value of 10 microM. Thus, thioredoxin is likely the reductant in vivo. Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in k(cat)/K(MetSO). Alternative mechanisms for sulfenic acid reduction are also presented based on analysis of available MsrA sequences.

International audience; Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. The proposed chemical mechanism ofEscherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 → Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.

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Background: Peptide methionine sulphoxide reductases catalyze the reduction of oxidized methionine residues in proteins. They are implicated in the defense of organisms against oxidative stress and in the regulation of processes involving peptide methionine oxidation/reduction. These enzymes are found in numerous organisms, from bacteria to mammals and plants. Their primary structure shows no significant similarity to any other known protein. Results: The X-ray structure of the peptide methionine sulphoxide reductase from Escherichia coli was determined at 3 A resolution by the multiple wavelength anomalous dispersion method for the selenomethionine-substituted enzyme, and it was refined to 1.9 A resolution for the native enzyme. The 23 kDa protein is folded into an α/β roll and contains a large proportion of coils. Among the three cysteine residues involved in the catalytic mechanism, Cys-51 is positioned at the N terminus of an α helix, in a solvent-exposed area composed of highly conserved amino acids. The two others, Cys-198 and Cys-206, are located in the C-terminal coil. Conclusions: Sequence alignments show that the overall fold of the peptide methionine sulphoxide reductase from E. coli is likely to be conserved in many species. The characteristics observed in the Cys-51 environment are in agreement with the expected accessibility of the active site of an enzyme that reduces methionine sulphoxides in various proteins. Cys-51 could be activated by the influence of an α helix dipole. The involvement of the two other cysteine residues in the catalytic mechanism requires a movement of the C-terminal coil. Several conserved amino acids and water molecules are discussed as potential participants in the reaction.

A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ∼7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer.